Migration of leukocytes from blood vessels into diseased tissues is crucial to the initiation of normal disease-fighting inflammatory responses. But this process, known as leukocyte recruitment, is also involved in the onset and progression of debilitating and life-threatening inflammatory and autoimmune diseases. The pathology of these diseases results from the attack of the body's immune system defenses on normal tissues. Thus, blocking leukocyte recruitment to target tissues in inflammatory and autoimmune disease would be a highly effective therapeutic intervention. The leukocyte cell classes that participate in cellular immune responses include lymphocytes, monocytes, neutrophils, eosinophils and mast cells. Lymphocytes are "master cells" that control the activity of most of these other cell types, particularly the monocytes. Lymphocytes are the leukocyte class that initiate, coordinate, and maintain the inflammatory response, and thus are the most important cells to block from entering inflammatory sites. Lymphocytes attract monocytes to the site, which are responsible for much of the actual tissue damage that occurs in inflammatory disease. Infiltration of these cells is responsible for a wide range of chronic, autoimmune diseases, and also organ transplant rejection. These diseases include rheumatoid arthritis, psoriasis, contact dermatitis, inflammatory bowel disease, multiple sclerosis, atherosclerosis, sarcoidosis, idiopathic pulmonary fibrosis, dermatomyositis, hepatitis, diabetes, allograft rejection, and graft-versus-host disease.
This process by which leukocytes leave the bloodstream and accumulate at inflammatory sites, and initiate disease, is best understood for neutrophils and monocytes, but is likely to be similar in broad outline for lymphocytes. This process takes place in at least three distinct steps (Springer, T. A., 1990, Nature 346:425-33; Lawrence and Springer, 1991, Cell 65:859-73; Butcher, E. C., 1991, Cell 67:1033-36). It is mediated at a molecular level by chemoattractant receptors, by cell-surface proteins called adhesion molecules, and by the ligands that bind to these two classes of cell-surface receptor. The major types of adhesion molecules are known as "selectins", "integrins" and "immunoglobulin (Ig) family" receptors.
Each of the three steps is essential for the emigration of the leukocytes to target tissues. Blocking the steps has been shown to prevent a normal inflammatory response, and impedes abnormal responses of inflammatory and autoimmune diseases (Harlan et al., 1992, In vivo models of leukocyte adherence to endothelium. In Adhesion: Its Role in Inflammatory Disease., J. M. Harlan and D. Y. Liu, (eds.), W. H. Freeman & Co., pp. 117-150). The steps of leukocyte adhesion and transendothelial migration can be summarized as follows:
Step 1. Primary adhesion. Leukocytes attach loosely to the blood vessel endothelium and "roll" slowly along the blood vessel wall, pushed by the flow of blood. Leukocyte-endothelium attachment is mediated by cell surface adhesion molecules called "selectins" which bind to carbohydrate-rich ligands ("glycoconjugates") on the leukocyte cell surface.
Step 2. Activation of leukocytes and migration to the target site. Chemoattractant receptors on the surface of the leukocytes bind chemoattractants secreted by cells at the site of damage or infection. Receptor binding activates the immune defenses of the leukocytes, and activates the adhesiveness of the adhesion molecules that mediate Step 3.
Step 3. Attachment and transendothelial migration. The leukocytes bind very tightly to the endothelial wall of the blood vessel and move to the junction between endothelial cells, where they begin to squeeze between these cells to reach the target tissue. This tighter binding is mediated by binding to adhesion receptors called "integrins" on the leukocytes to complementary receptors of the "Ig family" on the endothelium. (The Ig family molecules are named for their similarity to antibody molecules (immunoglobulins)). Chemoattractant receptors are also involved at this stage, as the leukocytes migrate up a concentration gradient of the chemoattractant secreted by cells at the target site.
These three classes of receptor-ligand interactions are all required and appear to act in a highly cooperative and coordinated manner to mediate leukocyte adherence to the microvasculature, diapedesis, and subsequent leukocyte mediated injury to tissue in inflammatory disease.
LFA-1 and Mac-1 together with p150,95 comprise the leukocyte integrins, a subfamily of integrins that share a common .beta. subunit (CD18) and have distinct .alpha.L, .alpha.M and .alpha.X (CD11a, b and c) .alpha. subunits (reviewed in Larson and Springer, 1990, Immunol. Rev. 114:181-217; Springer, 1990, Nature 346:425-433). They are required for leukocyte emigration as demonstrated by an absence of neutrophil extravasation (1) in patients with mutations in the common .beta. subunit (leukocyte adhesion deficiency), and (2) after treatment of healthy neutrophils with a monoclonal antibody (mAb) to the common .beta. subunit in vivo or in vitro (reviewed in Anderson and Springer, 1987, Ann. Rev. Med. 38:175-194; Larson and Springer, 1990, Immunol. Rev. 114:181-217).
The integrins LFA (lymphocyte function-associated antigen)-1 and Mac-1 on the neutrophil bind to the Ig family member ICAM (intercellular adhesion molecule)-1 on endothelium (Smith et al., 1988, J. Clin. Invest. 82:1746-1756; Smith et al., 1989, J. Clin. Invest. 83:2008-2017; Diamond et al., 1990, J. Cell Biol. 111:3129-3139). LFA-1 and not Mac-1 binds to ICAM-2 (de Fougerolles et al., 1991, J. Exp. Med. 174:253-267; Diamond et al., 1990, J. Cell Biol. 111:3129-3139), an endothelial cell molecule that is more closely related to ICAM-1 than these molecules are to other Ig superfamily members (Staunton et al., 1989, Nature 339:61-64).
The integrin VLA-4, that contains the .alpha.4 (CD49d) subunit noncovalently associated with the .beta.1 (CD29) subunit, is expressed by lymphocytes, monocytes, and neural crest-derived cells, and can interact with vascular cell adhesion molecule-1 (VCAM-1) (Elices et al., 1990, Cell 60:577). Like ICAM-1 and ICAM-2, VCAM-1 is a member of the Ig superfamily (Osborn et al., 1989, Cell 59:1203).
Chemoattractants are soluble mediators which activate cell adhesion and motility and direct cell migration through formation of a chemical gradient. They are produced by bacteria and numerous cell types including stimulated endothelial and stromal cells, platelets, tumor cells, cultured cell lines, and leukocytes themselves. The cells responding to chemoattractants appear to express specific receptors on their surfaces which bind the chemoattractant molecules and sense the gradient. Receptor stimulation induces cells to respond via a common signal transduction pathway which involves interaction of the chemoattractant-receptor complex with a guanine nucleotide or GTP-binding protein (G protein) (Gilman, A. G., 1987, Ann. Rev. Biochem. 56:615-49). This interaction stimulates phosphatidyl inositol hydrolysis by a phospholipase C, thus generating inositol phosphates and diacylglycerol. A transient rise in cytosolic free calcium then activates protein kinase C, and a variety of events including protein phosphorylation, membrane potential changes, and intracellular pH alterations ensue.
Several of the chemoattractants primarily affecting neutrophils were among the first chemoattractants identified. These include the complement component C5a, arachidonate derivative leukotriene B.sub.4 (LTB.sub.4), platelet activating factor (PAF), and formylmethionyl peptides of bacterial origin such as formyl-met-leu-phe (fMLP) (Devreotes and Zigmond, 1988, Annu. Rev. Cell Biol. 4:649-86). Although structurally dissimilar and stimulatory via separate receptors, these molecules produce a rapid and marked increase in neutrophil adhesiveness and motility leading to chemotaxis and prominent neutrophil accumulation in vivo (Pober and Cotran, 1990, Transplantation 50:537-44). The receptors for C5a and fMLP have been identified and sequenced; cDNA clones for each have also been generated (Gerard and Gerard, 1991, Nature 349:614-617; Boulay et al., 1990, Biophys. Res. Commun. 168:1103-09). These receptors share many structural features with one another and members of the "rhodopsin superfamily" of protein receptors (Dohlman et al., 1991, Ann. Rev. Biochem. 60:653-88).
More recently, a protein chemoattractant for neutrophils designated neutrophil activating protein-1 (NAP-1) or interleukin 8 (IL-8) was identified and molecularly cloned (Yoshimura et al., 1987, Proc. Natl. Acad. Sci. USA 84:9233-37; Matsushima et al., 1988, J. Exp. Med. 167:1883-93; Oppenheim et al., 1991, Annu. Rev. Immunol. 9:617-48). IL-8 was originally characterized as a 72 amino acid molecule produced by monocytes; variants of 79, 77, and 69 amino acids have subsequently been identified from additional sources including activated endothelial cells, lymphocytes, fibroblasts, and tumor lines. IL-8 has structural homology to a supergene family of novel 8-10 kDa cytokines isolated chiefly by subtractive hybridization (Oppenheim et al., 1991, Annu. Rev. Immunol. 9:617-48) and recently named the "chemokine" family. IL-8 and several other human cytokines, including platelet factor 4, platelet basic protein, IP-10, and melanoma growth stimulating factor/GRO comprise a subfamily of chemokines located on chromosome 4. In this subfamily, the relative positions of four cysteine residues are identical, with the first two cysteine residues separated by a single amino acid (C-X-C). Disulfide bonds between these four cysteines form two loops which appear to be essential for activity. The other subfamily, which includes the monocyte chemoattractants RANTES and monocyte chemoattractant protein-1 (see below), is clustered on chromosome 17, and the first two cysteines are adjacent (C-C).
The biological profile of activity for IL-8 is similar to that for C5a, LTB.sub.4, PAF, and fMLP; the respiratory burst is induced, neutrophil chemotaxis is promoted, and Mac-1 expression is increased on the surface of granulocytes (Baggiolini et al., 1989, J. Clin. Invest. 84: 1045-49; Detmers et al., 1990, J. Exp. Med. 171:1155-62). IL-8 differs from these others, however, in that it has been reported to attract approximately 10% of human peripheral blood T lymphocytes of either CD4.sup.+ or CD8.sup.+ subsets (Leonard et al., 1990, J. Immunol. 144:1323-30; Larsen et al., 1989, Science 241:1464-66), but does not attract monocytes. There is some controversy on whether IL-8 is a lymphocyte chemoattractant, because when injected in human skin it attracts neutrophils but not lymphocytes (Leonard et al, 1991, J. Invest. Dermatol. 96:690-94).
The chemoattractants which predominantly activate and guide monocytes include monocyte chemoattractant protein-1 (MCP-1) (Leonard and Yoshimura, 1990, Immunol. Today 11:97-101), the RANTES protein (Schall et al., 1990, Nature 347:669-71), and the neutrophil .alpha. granule protein CAP37 (Pereira et al., 1990, J. Clin. Invest. 85:1468-76), among others. As noted above, MCP-1 and RANTES are structurally homologous and belong to the subfamily of chemoattractive cytokines that are defined by a configuration of four cysteine residues in which the first two are adjacent (C-C). CAP37's structure is most homologous to proteins of the serine protease family (Peteira et al., 1990, J. Clin. Invest. 85:1468-76). The 76 amino acid MCP-1 is produced by activated endothelium, lymphocytes, macrophages, fibroblasts, smooth muscle cells, and tumor cells. It binds only to monocytes and induces approximately 30% of peripheral blood monocytes to respond in chemotaxis assays. The 68 amino acid RANTES is of special interest because it has been reported to selectively attract memory T helper cells (CD4.sup.+ and UCHL1 antigen/CD45RO positive) as well as monocytes. Of the chemoattractants known to attract lymphocytes, only RANTES appears subset-selective. However, the lymphocyte chemoattractive activity of RANTES may be weaker than its activity for eosinophils (Kameyoshi et al., 1992, J. Exp. Med. 176:587-92).
Endothelial cells cultured on matrices of type I collagen have been used to study neutrophil migration toward the chemoattractant IL-8 (Huber et al., 1991, Science 254:99-102). When tested with IL-8 contained in conditioned media from stimulated endothelial cells, neutrophils migrated similarly, and there was a similar signal-to-noise ratio, on collagen matrix plus endothelium and on matrix alone. Thus, there was no indication that the assay would be improved for other cell types by adding endothelium. Indeed, the expectation would have been that neutrophils and lymphocytes would behave similarly. Lymphocyte migration across endothelium into collagen gels has previously been reported in assays of migration (Kavanaugh et al., 1991, J. Immunol. 146:4149-4156; Masuyama et al., 1992, J. Immunol. 148:1367-1374). In this system, the endothelium is cultured on a collagen gel formed on a culture dish or well for several days, then lymphocytes are added. This is an assay of migration in which there is no evidence that chemotaxis is involved; rather, it appears to assay for a migratory subset of lymphocytes. There is no teaching of a chemoauractant. Furthermore, lymphocytes migrate into collagen gels even in the absence of an endothelial cell monolayer.
Compared with neutrophil and monocyte chemoattractants, little is known about chemoattractants for lymphocytes. The best characterized lymphocyte chemoattractants are RANTES and IL-8, which primarily attract monocytes and neutrophils, as noted above. Several in vitro studies have described lymphocyte chemotactic activities in the culture supernatants of mixed lymphocyte reactions and mitogen-stimulated human peripheral blood mononuclear cells (Cruikshank and Center, 1982, J. Immunol. 128:2569; Center and Cruikshank, 1982, J. Immunol. 128:2563-68; Van Epps et al., 1983, J. Immunol. 130:2727; Van Epps et al., 1983, J. Immunol. 131:687). One of these activities was related to a protein of apparent molecular weight 14 kD named lymphocyte chemotactic factor (LCF) (Cruikshank and Center, 1982, J. Immunol. 128:2569; Center and Cruikshank, 1982, J. Immunol. 128:2563-68) that was produced by T cells (Van Epps et al., 1983, J. Immunol. 130:2727). The initial purifications of LCF possibly yielded activities contaminated by the chemotactic mediators interleukin-1 and -2 (IL-1 and IL-2), however (Cruikshank and Center, 1982, J. Immunol. 128:2569). Subsequent purifications using high performance liquid chromatography have separated IL-1 and IL-2 from a LCF 10.5 kD in size (Potter and Van Epps, 1987, Cell. Immunol. 105:9-22). This LCF attracts lymphocytes, but selective subset chemotaxis was not assessed. Further characterization of the 10.5 kD LCF, including sequence information, has not been published. Another lymphocyte chemoattractant factor has been described that is 56,000 M.sub.r, binds to CD4, and is selectively chemoattractive for the CD4 subset of lymphocytes (Cruikshank et al., 1991, J. Immunol. 146:2928-34). It also is chemoattractive for monocytes (Cruikshank et al., 1991, J. Immunol. 146:2928-34) and eosinophils (Rand et al., 1991, J. Exp. Med. 173:1521-28). More recently, a less than 1 kD molecule that was extractable in lipid solvents (i.e., not a peptide) was isolated from normal human skin and named plasma-associated lymphocyte chemoattractant (Bacon et al., 1990, Eur. J. Immunol. 20:565-71). It has been suggested that this molecule is constitutively expressed in skin and accounts for surveillance lymphocyte trafficking there (Bacon et al., 1990, Eur. J. Immunol. 20:565-71). A variety of other agents or poorly characterized activities have also been reported. These include fetal calf serum, the protein casein, the mitogen phytohemagglutinin, and supernatants of stimulated peritoneal macrophages (Berman et al., 1988, Immunol. Invest. 17:625-77).
Although considerable effort has been invested on the study of lymphocyte chemoattractants, they remain poorly characterized relative to monocyte and neutrophil chemoattractants. Chemoattractants for the latter cell types, such as MCP-1 and IL-8, have been purified based on the conventional chemotaxis assay, sequenced, and cloned. However, no molecule identified primarily as a lymphocyte chemoattractive factor has been sequenced and cloned.
In large measure, the lack of rapid progress on lymphocyte chemoattractants appears due to the unreliability or lack of biological relevance of currently available lymphocyte chemotaxis assays. For example, the protein casein, fetal calf serum, the mitogen phytohemagglutinin, and the hormone insulin, have all been reported active in lymphocyte chemotaxis assays (Berman et al., 1988, Immunol. Invest. 17:625-77), but the first three are not physiologic chemoattractants, and it seems doubtful that insulin is, because lymphocytes do not accumulate at sites of insulin injection in diabetics. Currently available lymphocyte chemotaxis assays have been reviewed (Berman et al., 1988, Immunol. Invest. 17:625-77). The most widely used is the Boyden Chamber assay, in which a microporous membrane divides two chambers, the lower containing the test chemoattractant and the upper containing the cells, e.g. lymphocytes. The microporous membrane is commonly nitrocellulose or polycarbonate, and may be coated with a protein such as collagen. The distance of migration into the filter, the number of cells crossing the filter that remain adherent to the undersurface, or the number of cells that accumulate in the lower chamber may be counted.